CN107202836B - Method for rapidly analyzing theanine content in fresh tea sample - Google Patents
Method for rapidly analyzing theanine content in fresh tea sample Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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Abstract
A method for rapidly analyzing the content of theanine in a fresh tea sample is characterized by comprising the following steps: crushing and uniformly mixing fresh tea leaves, extracting for 20min at 90 ℃ by using ultrapure water as an extraction solvent, performing centrifugal filtration, performing pre-column derivatization by using 6-aminoquinoline-N-hydroxysuccinimide carbamate as a derivatization agent, performing gradient elution by using an Xbridge C18 chromatographic column, and performing separation and detection by using a high performance liquid chromatography-fluorescence detector. The method has the advantages of rapid and simple sample preparation, low cost, good precision, accuracy, stability and linear relation, rapid and sensitive whole analysis process and good reproducibility, and is suitable for rapid analysis of theanine in fresh tea samples.
Description
Technical Field
The invention relates to the field of analytical chemistry, in particular to a rapid analysis method for theanine content in a fresh tea sample.
Background
The tea is one of three natural beverages in the world, has unique fragrance and rich nutrition and health care efficacy, and is popular with consumers. Tea leaves contain abundant amino acids, which are important components constituting the taste of tea leaves and directly affect the quality of tea leaves. Wherein, the theanine is a specific amino acid in the tea and accounts for more than 50 percent of the total amino acid content of the tea. The theanine is safe and nontoxic, has various biological functions of reducing blood pressure, resisting fatigue, resisting tumors and the like, and is widely used in the fields of medical treatment, health care, food, beverage, fine chemical engineering and the like along with the discovery of the physiological function and the medical value of the theanine. The accurate quantitative analysis of theanine has very important significance for the evaluation of the quality of tea, the application research and development of theanine, the research of the functional metabolism and the like.
At present, in the research of the method for analyzing theanine in tea, dry samples are more and more mature, and as specified in the national standard GB/T8303-2013, the tea samples need to be ground and then heated in an electrothermal constant-temperature drying oven, and the ground tea samples are used for measuring the content of amino acid after water is removed to constant weight. Because the dry sample is usually prepared by high-temperature enzyme deactivation, drying and crushing, the protein is denatured in the high-temperature process, the biological activity of protease is lost, the protein is easily degraded to cause the increase of the content of amino acid, and meanwhile, the steps of drying and the like can cause the conversion of various biochemical components of the tea leaves, so that the measured data can not reflect the real level of the amino acid in the tea leaves, thereby generating detection errors.
The method for analyzing theanine in tea mainly comprises ninhydrin colorimetric method, gas chromatography and mass spectrum combination method thereof, capillary electrophoresis method, high performance liquid chromatography and the like. Classical analytical methods typically employ an amino acid analyzer using ninhydrin as the derivatizing reagent for post-column derivatization assays. However, the amino acid analyzer is expensive, long in analysis time and strong in specificity, can only be used for analyzing free amino acids such as theanine and the like, and limits the wide application of the amino acid analyzer. Compared with other methods, the pre-column derivatization-high performance liquid chromatography does not need a special reaction device, has the advantages of high instrument popularization rate, short analysis time, flexible and various methods, high sensitivity and easy popularization, and gradually becomes a conventional means for detecting theanine. The invention patent of China 'method for detecting free amino acids in tea by using reversed-phase high performance liquid chromatography' (patent number ZL 201510109474.4) takes 6-aminoquinoline-N-hydroxysuccinimidyl carbamate as a pre-column derivatization agent, uses an amino acid special analysis column for gradient elution, and combines the reversed-phase high performance liquid chromatography to realize quantitative analysis of 19 amino acids such as theanine and the like in tea. However, the method adopts the chromatographic column special for amino acid separation, needs complex gradient separation, has high use cost and long operation period (about 1 h), takes time in the analysis process of a large amount of samples, and is not beneficial to rapid analysis and popularization of theanine. More importantly, the method does not have necessary methodology tests on the accuracy, sensitivity, precision and the like of the method, and the technical effect cannot be effectively guaranteed.
Disclosure of Invention
The invention provides a method for rapidly analyzing the content of theanine in a fresh tea sample, and aims to solve the problems of low speed, low efficiency, high cost, difficult basic application and popularization and the like in the existing method for analyzing the theanine.
In order to achieve the purpose, the invention adopts the technical scheme that: a method for rapidly analyzing the content of theanine in a fresh tea sample comprises the following two parts:
preparing a solution, and establishing a standard curve of the tested theanine with known gradient concentration by using a high performance liquid chromatography-fluorescence detection method; the establishment of the standard curve comprises the following steps:
step (1), preparing a theanine standard substance, and then respectively preparing 0.14 mol/L acetate buffer solution, 0.4mol/L borate buffer solution, 1mg/mL AQC derivative solution and 6 kinds of theanine standard working solutions with concentrations of 1 mu mol/L, 5 mu mol/L, 50 mu mol/L, 500 mu mol/L, 1000 mu mol/L and 1250 mu mol/L;
step (2), performing derivatization reaction on the theanine standard working solution;
transferring the theanine standard working solutions with 6 concentrations, putting 10 mu L of the theanine standard working solution with each concentration into an automatic sampling bottle, respectively adding 70 mu L of the borate buffer solution, and mixing in a vortex manner; respectively taking 15 mu L of AQC derivative liquid and 5 mu L of acetonitrile, adding the AQC derivative liquid and the acetonitrile into an automatic sample injection bottle in a vortex state, carrying out vortex mixing, standing, heating at 50-55 ℃ for 8-15 min, taking out, and cooling to room temperature for analysis;
step (3), determining the chromatographic peak retention time and the chromatographic peak area of theanine in the derivatized theanine standard working solution by using a high performance liquid chromatography-fluorescence detection method, determining the nature by using the chromatographic peak retention time, and then drawing the standard curve by using the molar concentration of the theanine standard working solution as a horizontal coordinate and using the chromatographic peak area as a vertical coordinate;
wherein, the separation conditions of the instrument are as follows:
a chromatographic column: an Xbridge C18 column (specification 3.9 mm. times.15 cm, 4 μm); the column temperature is 37 ℃; the flow rate is 2.0 mL/min;
fluorescence detection: excitation wavelength of 250nm and emission wavelength of 395 nm;
mobile phase: a is acetate buffer solution which is diluted by ultrapure water according to the volume ratio of l to 10; b is 100% acetonitrile; c is 100% ultrapure water; gradient elution procedure: 0min, 100% A; 0.5min, 98% A + 2.0B%; 0.5-9.0min, 96.5% A +3.5% B; 9.0-9.5min, 95.0% A +5.0% B; 9.5-11.5min, 91.5% A + 8.5% B; 11.5-13.0min, 83.0% A +17.0% B, keeping for 4 min; 17.0min, 60.0% B +40% C, keeping for 2 min; 19-23min, 100% A; the sample volume is 10 mu L;
the second part, the content of theanine in the first part in the fresh tea sample is measured, and the method comprises the following steps:
step (1), preparing a sample;
taking a fresh tea sample, crushing and uniformly mixing, adding boiling water into the fresh tea sample, wherein the adding ratio of the fresh tea sample to the boiling water is that 40mL of boiling water is added into each 1g of fresh tea sample, leaching for 18-22 min at 88-92 ℃, cooling to room temperature, centrifuging for 4-6 min at 2500-3500 r/min, adding water into supernatant to a constant volume of 10mL, and filtering through a 0.45-micrometer microporous filter membrane to obtain a fresh tea sample solution;
step (2), carrying out derivatization reaction on the fresh tea leaf sample solution;
transferring 10 mu L of fresh tea leaf sample solution, placing the fresh tea leaf sample solution in an automatic sample feeding bottle, adding 70 mu L of borate buffer solution, and mixing by vortex; respectively taking 15 mu L of AQC derivative liquid and 5 mu L of acetonitrile, adding the AQC derivative liquid and the acetonitrile into an automatic sample injection bottle in a vortex state, carrying out vortex mixing, standing, heating at 50-55 ℃ for 8-15 min, taking out, and cooling to room temperature for analysis;
step (3), determining the quality of the theanine in the fresh tea leaf sample solution according to the retention time of the chromatographic peak of the theanine in the standard working solution of the theanine, and calculating the content of the theanine in the fresh tea leaf sample solution by using an external standard curve method; wherein the separation conditions of the instrument for measuring the theanine in the fresh tea leaf sample solution are the same as those of the instrument in the step (3) of the first part.
The relevant content in the above technical solution is explained as follows:
1. in the above scheme, in the step (1) of the first part, the preparation method of 0.14 mol/L acetate buffer solution is as follows: weighing 19.0g of sodium acetate trihydrate and 1.72g of triethylamine, dissolving the sodium acetate trihydrate and the 1.72g of triethylamine in 1000mL of water, adjusting the pH value to 5.05 by using phosphoric acid, adding EDTA, and filtering by using a 0.45 mu m filter membrane;
the preparation method of 0.4mol/L borate buffer solution comprises the following steps: weighing 12.36g of boric acid, adding 400mL of water for dissolving, adjusting the pH to 8.8 by using 400g/L of sodium hydroxide solution, and then adding water for diluting to 500 mL;
the preparation method of the 1mg/mL AQC derivative solution comprises the following steps: to 1mg of AQC powder, 1mL of acetonitrile was added, mixed by vortexing, and heated to dissolve at 55 ℃. AQC means 6-aminoquinolyl-N-hydroxysuccinimidyl formate, produced by Waters corporation, acetonitrile is chromatographically pure.
Preparing a theanine standard mother solution: precisely weighing a proper amount of theanine standard substance, placing the theanine standard substance in a 25mL volumetric flask, adding ultrapure water for dissolving, and fixing the volume to scale to obtain a theanine standard solution with the concentration of 12.5 mu mol/L, and storing the theanine standard solution in a refrigerator at the temperature of-20 ℃. And preparing the standard working solution of the theanine with the 6 concentrations by using the standard mother solution of the theanine.
The working principle and the beneficial effects of the invention are as follows: aiming at the problems of low speed, low efficiency, high cost, difficult popularization and the like in the existing theanine analysis technology, the invention establishes a rapid, practical, efficient and accurate method for separating and analyzing theanine in a fresh tea sample through technical innovation. Crushing and uniformly mixing fresh tea leaves, extracting for 20min at 90 ℃ by using ultrapure water as an extraction solvent, performing centrifugal filtration, performing pre-column derivatization by using 6-aminoquinoline-N-hydroxysuccinimide carbamate as a derivatization agent, performing gradient elution by using an Xbridge C18 chromatographic column, and performing separation and detection by using a high performance liquid chromatography-fluorescence detector. In the traditional method, a tea dry sample is used as a sample to detect the content of theanine, because a tea fresh sample contains more interference substances such as moisture, pigments, water-soluble ash, tea polyphenol and the like than the tea dry sample, the subsequent separation is easy to cause difficulty, namely, the existing detection method for detecting the theanine in the tea dry sample is not suitable for the detection of the tea fresh sample. According to the invention, a fresh tea sample is used as a sample instead of a dry tea sample, in order to solve the difficult separation problem, 6-aminoquinoline-N-hydroxysuccinimide carbamate is creatively and pertinently selected as a pre-column derivatization agent, and separation and analysis are carried out by combining an Xbridge C18 chromatographic column and a high performance liquid chromatography-fluorescence detector, so that the theanine in the fresh tea sample can be quickly, efficiently and accurately separated and analyzed. Particularly, a high-efficiency Xbridge C18 chromatographic column with the specification of 4 mu m is selected and combined with an AQC pre-column derivatization method, so that the theanine in the fresh tea sample can be well separated while the analysis speed is ensured to be higher, and the qualitative and quantitative analysis of the theanine in the fresh tea sample is ensured.
Compared with the prior art for analyzing theanine, the method has the beneficial effects that:
①, a rapid quantitative analysis method for theanine in a fresh tea sample is established for the first time, and the method has the advantages that the fresh tea sample is used as the sample, the detection of the theanine content by using a dry tea sample as the sample in the traditional method is replaced, the detection errors caused by the conversion of various biochemical components of the tea and the change of the theanine content possibly caused by the steps of high temperature, drying and the like are avoided, the measurement data is more accurate and reliable, and the content level of the theanine in the tea can be truly reflected;
② selecting XBridge C18 chromatographic column to replace amino acid special analytical column, separating and analyzing theanine, through technical optimization, the whole analysis period is only 14 minutes, greatly improving the analysis efficiency of theanine, satisfying the requirement of large batch sample analysis to progress, at the same time, XBridge C18 chromatographic column is relatively cheap, has weak specificity, can be used for the detection of other substances, under the premise of saving analysis cost, makes up the vacancy that the theanine in tea can not be accurately and rapidly analyzed based on high performance liquid chromatography at present, is especially suitable for being popularized and used in vast base level detection mechanism and units.
In a word, the sample preparation of the invention is rapid and simple, the cost is low, the precision, the accuracy, the stability and the linear relation are good, the whole analysis process is rapid, sensitive and good in reproducibility, the invention is suitable for rapid analysis of theanine in fresh tea samples, an effective analysis method is provided for quality control of theanine in tea, and the invention is suitable for popularization and application.
Drawings
FIG. 1 is an HPLC chromatogram of a standard working solution of theanine of the present invention.
Detailed Description
The invention is further described with reference to the following figures and examples:
example (b): method for rapidly analyzing theanine content in fresh tea sample
The rapid analysis method comprises the following two parts:
preparing a solution, and establishing a standard curve of the tested theanine with known gradient concentration by using a high performance liquid chromatography-fluorescence detection method; the establishment of the standard curve comprises the following steps:
step (1), preparing a theanine standard substance, and then respectively preparing 0.14 mol/L acetate buffer solution, 0.4mol/L borate buffer solution, 1mg/mL AQC derivative solution and 6 kinds of theanine standard working solutions with concentrations of 1 mu mol/L, 5 mu mol/L, 50 mu mol/L, 500 mu mol/L, 1000 mu mol/L and 1250 mu mol/L;
the preparation method of 0.14 mol/L acetate buffer solution comprises the following steps: weighing 19.0g of sodium acetate trihydrate and 1.72g of triethylamine, dissolving the sodium acetate trihydrate and the 1.72g of triethylamine in 1000mL of water, adjusting the pH value to 5.05 by using phosphoric acid, adding EDTA, and filtering by using a 0.45 mu m filter membrane;
the preparation method of 0.4mol/L borate buffer solution comprises the following steps: weighing 12.36g of boric acid, adding 400mL of water for dissolving, adjusting the pH to 8.8 by using 400g/L of sodium hydroxide solution, and then adding water for diluting to 500 mL;
the preparation method of the 1mg/mL AQC derivative solution comprises the following steps: to 1mg of AQC powder, 1mL of acetonitrile was added, mixed by vortexing, and heated to dissolve at 55 ℃.
Preparing a theanine standard mother solution: precisely weighing a proper amount of theanine standard substance, placing the theanine standard substance in a 25mL volumetric flask, adding ultrapure water for dissolving, and fixing the volume to scale to obtain a theanine standard solution with the concentration of 12.5 mu mol/L, and storing the theanine standard solution in a refrigerator at the temperature of-20 ℃. And preparing the standard working solution of the theanine with the 6 concentrations by using the standard mother solution of the theanine.
Preparing an instrument and equipment: 2695 high performance liquid chromatograph equipped with 2475 fluorescence detector (Waters corporation); TG16-WS desk-top high-speed centrifuge (Hunan instruments laboratory Ltd.); a K600 pulverizer (bolan, germany); LE-3000 electric constant temperature water bath (Shanghai leap into medical instruments Co.); Direct-Q5 UV ultra-pure water machine (Millipore, USA).
Step (2), performing derivatization reaction on the theanine standard working solution;
transferring the theanine standard working solutions with 6 concentrations, putting 10 mu L of the theanine standard working solution with each concentration into an automatic sampling bottle, respectively adding 70 mu L of the borate buffer solution, and mixing in a vortex manner; respectively taking 15 mu L of AQC derivative liquid and 5 mu L of acetonitrile, adding the AQC derivative liquid and the acetonitrile into an automatic sample injection bottle in a vortex state, carrying out vortex mixing for 10-20 s, standing for 1min, heating at 55 ℃ for 10min, taking out, and cooling to room temperature for analysis;
step (3), determining the chromatographic peak retention time and the chromatographic peak area of theanine in the derivatized theanine standard working solution by using a high performance liquid chromatography-fluorescence detection method, determining the nature by using the chromatographic peak retention time, and then drawing the standard curve by using the molar concentration of the theanine standard working solution as a horizontal coordinate and using the chromatographic peak area as a vertical coordinate;
wherein, the separation conditions of the instrument are as follows:
a chromatographic column: an Xbridge C18 column (3.9 mm. times.15 cm, 4 μm, Waters Corp.); the column temperature is 37 ℃; the flow rate is 2.0 mL/min;
fluorescence detection: excitation wavelength of 250nm and emission wavelength of 395 nm;
mobile phase: a is acetate buffer solution which is diluted by ultrapure water according to the volume ratio of l to 10; b is 100% acetonitrile; c is 100% ultrapure water; gradient elution procedure: 0min, 100% A; 0.5min, 98% A + 2.0B%; 0.5-9.0min, 96.5% A +3.5% B; 9.0-9.5min, 95.0% A +5.0% B; 9.5-11.5min, 91.5% A + 8.5% B; 11.5-13.0min, 83.0% A +17.0% B, keeping for 4 min; 17.0min, 60.0% B +40% C, keeping for 2 min; 19-23min, 100% A; the sample volume is 10 mu L;
the second part, the content of theanine in the first part in the fresh tea sample is measured, and the method comprises the following steps:
step (1), preparing a sample;
taking a fresh tea sample, crushing and uniformly mixing, weighing 0.25g, adding 10mL of boiling water, leaching for 20min in a water bath kettle at 90 ℃, cooling to room temperature, centrifuging for 5min at 3000r/min, adding water into supernatant to a constant volume of 10mL, and filtering through a 0.45-micron microporous filter membrane to obtain a fresh tea sample solution for later use;
step (2), carrying out derivatization reaction on the fresh tea leaf sample solution;
transferring 10 mu L of fresh tea leaf sample solution, placing the fresh tea leaf sample solution in an automatic sample feeding bottle, adding 70 mu L of borate buffer solution, and mixing by vortex; respectively taking 15 mu L of AQC derivative liquid and 5 mu L of acetonitrile, adding the AQC derivative liquid and the acetonitrile into an automatic sample injection bottle in a vortex state, carrying out vortex mixing for 10-20 s, standing for 1min, heating at 55 ℃ for 10min, taking out, and cooling to room temperature for analysis;
step (3), determining the quality of the theanine in the fresh tea leaf sample solution according to the retention time of the chromatographic peak of the theanine in the standard working solution of the theanine, and calculating the content of the theanine in the fresh tea leaf sample solution by using an external standard curve method; wherein the separation conditions of the instrument for measuring the theanine in the fresh tea leaf sample solution are the same as those of the instrument in the step (3) of the first part.
Test results of this example:
1. chromatographic separation of theanine standard working solution
As can be seen from the attached figure 1, the peak time of the standard working solution of theanine is about 11min after the derivation, and no other interference exists before and after the peak, which indicates that the method can be used for accurate qualitative and quantitative determination of theanine.
2. Regression equation, correlation coefficient and detection limit of method
Preparing theanine standard solutions with concentrations of 1, 5, 50, 500, 1000 and 1250 [ mu ] mol/L, respectively, performing derivatization, and determining by sample injection to obtain theanine solution concentration (L) ((L))X) As abscissa, corresponding peak area: (Y) For the ordinate, a standard working curve was plotted and subjected to linear regression analysis to calculate the correlation coefficient (table 1).
The result shows that the linear relation between the concentration of theanine and the peak area thereof is good within the range of 5-250 mu mol/L, and the correlation coefficient is 0.9991. The detection limit of the calculation method by 3 times of the signal-to-noise ratio is 0.05 mu mol/L.
TABLE 1 Linear equation, correlation coefficient and detection limit for theanine
3. Method recovery
Precisely weighing 5 parts of 0.25g of fresh tea leaf sample with known theanine content, adding a certain volume of standard solution, performing sample preparation and derivatization reaction, measuring, and quantifying by an external standard method to obtain the average recovery rate of the theanine of 87.91 and the RSD of 9.02. The method has the advantages of high accuracy, good reproducibility and reliability.
4. Precision of the method
Taking a proper amount of standard solution containing theanine, performing sample injection analysis after derivation according to the method, continuously performing sample injection for 5 times, calculating RSD by using retention time and peak area of chromatographic peak as indexes, and inspecting precision. The obtained theanine has the retention time RSD of 0.49 percent and the peak area RSD of 1.97 percent, which indicates that the method has higher precision.
5. Method repeatability
5 parts of the same fresh tea leaf sample is taken, extracted, derived and measured according to the method, the RSD is respectively calculated by taking the retention time and the peak area of a chromatographic peak as indexes, and the repeatability of the method is inspected. The retention time RSD of the theanine peak contained in the fresh tea leaves is 1.68%, the peak area RSD is 2.55, and the repeatability is good.
6. Method stability
And (3) taking the same fresh tea leaf sample as a test solution, deriving according to the method, injecting samples for 0, 4, 8, 12 and 24 hours respectively, calculating RSD by taking retention time and peak area of chromatographic peaks as indexes, and inspecting the stability of the fresh tea leaf derivative solution. The theanine-derived product retention time RSD was 1.50% (n =5) and the peak area RSD was 2.54% (n = 5). Showing that the amino acid derivatization solution can be stable for 24 hours at room temperature.
7. Application of the method
Using the established method, theanine was measured in 2 fresh samples of Biluochun tea taken from Dongtong mountain and West mountain of Dongting, Suzhou, respectively, and the results are shown in table 2.
TABLE 2 detection results of theanine in fresh tea samples
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (2)
1. A method for rapidly analyzing the content of theanine in a fresh tea sample is characterized by comprising the following steps: the rapid analysis method comprises the following two parts:
preparing a solution, and establishing a standard curve of the tested theanine with known gradient concentration by using a high performance liquid chromatography-fluorescence detection method; the establishment of the standard curve comprises the following steps:
step (1), preparing a theanine standard substance, and then respectively preparing 0.14 mol/L acetate buffer solution, 0.4mol/L borate buffer solution, 1mg/mL AQC derivative solution and 6 kinds of theanine standard working solutions with concentrations of 1 mu mol/L, 5 mu mol/L, 50 mu mol/L, 500 mu mol/L, 1000 mu mol/L and 1250 mu mol/L; the preparation method of 0.14 mol/L acetate buffer solution comprises the following steps: weighing 19.0g of sodium acetate trihydrate and 1.72g of triethylamine, dissolving the sodium acetate trihydrate and the 1.72g of triethylamine in 1000mL of water, adjusting the pH value to 5.05 by using phosphoric acid, adding EDTA, and filtering by using a 0.45 mu m filter membrane;
step (2), performing derivatization reaction on the theanine standard working solution;
transferring the theanine standard working solutions with 6 concentrations, putting 10 mu L of the theanine standard working solution with each concentration into an automatic sampling bottle, respectively adding 70 mu L of the borate buffer solution, and mixing in a vortex manner; respectively taking 15 mu L of AQC derivative liquid and 5 mu L of acetonitrile, adding the AQC derivative liquid and the acetonitrile into an automatic sample injection bottle in a vortex state, carrying out vortex mixing, standing, heating at 50-55 ℃ for 8-15 min, taking out, and cooling to room temperature for analysis;
step (3), determining the chromatographic peak retention time and the chromatographic peak area of theanine in the derivatized theanine standard working solution by using a high performance liquid chromatography-fluorescence detection method, determining the nature by using the chromatographic peak retention time, and then drawing the standard curve by using the molar concentration of the theanine standard working solution as a horizontal coordinate and using the chromatographic peak area as a vertical coordinate;
wherein, the separation conditions of the instrument are as follows:
a chromatographic column: an Xbridge C18 chromatographic column with the specification of 3.9mm multiplied by 15cm and 4 μm; the column temperature is 37 ℃; the flow rate is 2.0 mL/min;
fluorescence detection: excitation wavelength of 250nm and emission wavelength of 395 nm;
mobile phase: a is acetate buffer solution which is diluted by ultrapure water according to the volume ratio of l to 10; b is 100% acetonitrile; c is 100% ultrapure water; gradient elution procedure: 0min, 100% A; 0.5min, 98% A + 2.0B%; 0.5-9.0min, 96.5% A +3.5% B; 9.0-9.5min, 95.0% A +5.0% B; 9.5-11.5min, 91.5% A + 8.5% B; 11.5-13.0min, 83.0% A +17.0% B, keeping for 4 min; 17.0min, 60.0% B +40% C, keeping for 2 min; 19-23min, 100% A; the sample volume is 10 mu L;
the second part, the content of theanine in the first part in the fresh tea sample is measured, and the method comprises the following steps:
step (1), preparing a sample;
taking a fresh tea sample, crushing and uniformly mixing, adding boiling water into the fresh tea sample, wherein the adding ratio of the fresh tea sample to the boiling water is that 40mL of boiling water is added into each 1g of fresh tea sample, leaching for 18-22 min at 88-92 ℃, cooling to room temperature, centrifuging for 4-6 min at 2500-3500 r/min, adding water into supernatant to a constant volume of 10mL, and filtering through a 0.45-micrometer microporous filter membrane to obtain a fresh tea sample solution;
step (2), carrying out derivatization reaction on the fresh tea leaf sample solution;
transferring 10 mu L of fresh tea leaf sample solution, placing the fresh tea leaf sample solution in an automatic sample feeding bottle, adding 70 mu L of borate buffer solution, and mixing by vortex; respectively taking 15 mu L of AQC derivative liquid and 5 mu L of acetonitrile, adding the AQC derivative liquid and the acetonitrile into an automatic sample injection bottle in a vortex state, carrying out vortex mixing, standing, heating at 50-55 ℃ for 8-15 min, taking out, and cooling to room temperature for analysis;
step (3), determining the quality of the theanine in the fresh tea leaf sample solution according to the retention time of the chromatographic peak of the theanine in the standard working solution of the theanine, and calculating the content of the theanine in the fresh tea leaf sample solution by using an external standard curve method; wherein the separation conditions of the instrument for measuring the theanine in the fresh tea leaf sample solution are the same as those of the instrument in the step (3) of the first part.
2. The method for rapidly analyzing the content of theanine in the fresh tea sample according to claim 1, which is characterized in that: in the first step (1), 0.4mol/L borate buffer solution is prepared by the following method: weighing 12.36g of boric acid, adding 400mL of water for dissolving, adjusting the pH to 8.8 by using 400g/L of sodium hydroxide solution, and then adding water for diluting to 500 mL;
the preparation method of the 1mg/mL AQC derivative solution comprises the following steps: to 1mg of AQC powder, 1mL of acetonitrile was added, mixed by vortexing, and heated to dissolve at 55 ℃.
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| CN108007878A (en) * | 2017-11-10 | 2018-05-08 | 桂林电子科技大学 | A kind of portable theanine detecting instrument and detection method |
| CN107796772A (en) * | 2017-11-10 | 2018-03-13 | 桂林电子科技大学 | A kind of portable instrument and the method for detecting theanine |
| CN111393539A (en) * | 2020-05-22 | 2020-07-10 | 陕西理工大学 | Preparation method for co-producing tea polysaccharide, theanine and caffeine in summer and autumn tea |
| CN112326855A (en) * | 2020-11-10 | 2021-02-05 | 胡毅 | Method for rapidly measuring 12 water extracts of tea |
| CN112557532A (en) * | 2020-11-27 | 2021-03-26 | 北京康立生医药技术开发有限公司 | Detection method of composition containing theanine |
| CN115015446B (en) * | 2022-04-18 | 2024-03-08 | 中华人民共和国日照海关 | Method for simultaneously measuring L-theanine, caffeine, tea polyphenol and hexadecanoic acid in green tea |
| CN114965745A (en) * | 2022-04-21 | 2022-08-30 | 武汉轻工大学 | Method for detecting amino acids in tea |
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